Ectopic expression of sericin enables efficient production of ancient silk with structural changes in silkworm

Bombyx mori silk is a super-long natural protein fiber with a unique structure and excellent performance. Innovative silk structures with high performance are in great demand, thus resulting in an industrial bottleneck. Herein, the outer layer sericin SER3 is ectopically expressed in the posterior silk gland (PSG) in silkworms via a piggyBac-mediated transgenic approach, then secreted into the inner fibroin layer, thus generating a fiber with sericin microsomes dispersed in fibroin fibrils. The water-soluble SER3 protein secreted by PSG causes P25’s detachment from the fibroin unit of the Fib-H/Fib-L/P25 polymer, and accumulation between the fibroin layer and the sericin layer. Consequently, the water solubility and stability of the fibroin-colloid in the silk glandular cavity, and the crystallinity increase, and the mechanical properties of cocoon fibers, moisture absorption and moisture liberation of the silk also improve. Meanwhile, the mutant overcomes the problems of low survival and abnormal silk gland development, thus enabling higher production efficiency of cocoon silk. In summary, we describe a silk gland transgenic target protein selection strategy to alter the silk fiber structure and to innovate its properties. This work provides an efficient and green method to produce silk fibers with new functions.

Innovative silkworm silk structures with higher fiber performance are in great demand.
In the manuscript entitled "Ectopic expression of sericin enables efficient production of ancient silk with structural changes in silkworm", the authors Chen et al. have ectopically expressed the outer layer sericin SER3 in the PSG of the silkworm by a piggyBac-mediated transgenic approach, thus generating a new fiber with improved β-sheet structure contents and mechanical properties, moisture absorption and moisture liberation properties. They found that the transgenic silkworm varieties have higher cocoon production efficiency without affecting silk gland development. Hence, they concluded it is an efficient, green method to produce new silk fibers with innovative properties via the silk gland transgenic target protein selection strategy.
The topic of this paper is very interesting. The authors have presented the observation and analysis after ectopic expression of SER3 in the PSG using TEM, FTIR etc.
Several aspects of this paper -especially a more complete description and discussion of the method used and the results-could be improved including:

1) About novelty
Several previous studies have reported strategies to affect the mechanical properties of silk by overexpressing a specific protein (SER3) in the fibrin layer of silk fibers (see Refs. 23-28, provided by the authors). Although they state that SER3 is an endogenous protein specifically expressed in the MSG, but not in the PSG of the silkworm. SER3 should be considered as a foreign protein of the PSG. Therefore, it is not the first report on the use of exogenous proteins to modify the properties of silk fiber, and the results are predictable.

2) Insufficient analysis of mechanisms
1. In Fig. 2d, the authors have observed the separation of P25 from fibroin and the location of P25 between fibroin and sericin after expression of SER3 in the PSG.
It is a very interesting result. How did SER3 change the component and structure of fibroin? The structure and properties of SER3 and whether it interacts with Fib-H/Fib-L need to have a comparative analysis and a reasonable analysis of the experimental results is required.
2. As the authors have shown that SER3 has cysteine residue, why did SER3 not interact with Fib-H or Fib-L via disulfide bonds instead of an independent SM formation?
3. Why was P25 located between fibroin and sericin? 4. How did P25 affect the stability and structure of fibroin? 5. The authors have shown that SM is free or independent from fibroin. SER3 is predicted to have α-helix structure without β-sheet. How did SER3 expression result in an increase in β-sheet content of silk fiber? 6. Usually, the amorphous region formed by α-helix and random coil structure determines the elasticity and strain of animal silk. It is doubtful that the decrease in α-helix content will not affect the strain (Fig. 3c). 7. Line 256, "Moreover, the sericin microsomes dispersed in the fibrils significantly improved the moisture absorption and liberation of the silk fibers, thereby improving the performance of the textile material." In fact, the sericin microsomes were dispersed in the fibrils, which may disrupt the structure of mutant silk. How does it improve the mechanical properties of mutant silk?

3) Technical and methodological issues
1. In Fig. 1b/2b, more evidence such as MS, WB should be provided to claim SM observed in the fibrils. Fig. 2a, an image of silk fiber under white light should be provided to clearly indicate the details of silk fiber.

In
3. In Fig. 2c, it is undoubtful that the sericin content in silk layer increased as SER3 was overexpressed in the PSG and then secreted into silk fiber. The authors should measure the expression of SER3 in the PSG and the content of SER3, Fib-H, Fib-L and P25 in the silk fiber to better support the claim of high silk yield.
However, we also noticed that there is no significant difference in cocoon weight ( Fig. S2i) and an increase in the cocoon layer ratio (Fig. S2j), which indicated a decrease in the pupal weight of SER3 silkworm. Hence, the high silk yield via ectopic expression of SER3 in the PSG is not convincing. 4. In Fig. 2c, the authors stated that the percentage of sericin in cocoon silk in the SER group was 7.39% higher than that in the WT group, an increase in 21.8%.
How did the authors calculate the percentage of sericin in cocoon in SER3 group and make the comparison with WT group? I suggest they perform SDS-PAGE and western blot to clearly show the expression of SER3 in the fibroin layer, but not the actual yield of silk protein. 5. In Fig. 2e-f, the result is not convincing as different peak numbers were applied for the curve-fitting and the determination of the secondary structure. Also, the same wavenumber was assigned to different secondary structure. In fact, according to the data provided by the authors, the curve-fitting results using a general procedure showed that the β-sheet content was 42.05% in the WT group and 44.38% in the SER3 group. The difference between the two groups did not appear to be significant. 6. The authors should provide more evidence that the silk structure has been indeed changed. I suggest to perform SAXS or WAXS for characterizing silk crystal structure and size. 7. As shown in Fig. 4c, it is strange that Fib-H/L/P25 appeared in MP. I suggest the authors should take care the part of silk gland for PCR. Also, there is a significant difference between the relative expression of EGFP and SER3. Could the authors explain the difference in the expression of EGFP and SER3 since they are fused together?
8. According to the results provided by the authors, the silk structure of the mutant was changed dramatically. Then the author should analyze the microstructure of mutant silk more comprehensively, as FTIR is usually considered as a semiquantitative method. Wide-angle X-ray diffraction or small-angle X-ray scattering could clearly characterize the crystallinity, grain size, orientation and other key structural information of polymer materials, which should be supplemented to better indicate the structural changes of mutant silk. 9. It is suggested that proteomic analysis of degumming mutant silk should be performed to precisely determine SER3 content in silk fiber, which is essential to address the mechanism by which SER3 affects the structure and properties of silk. 10. The author mentioned that the number of cocoons for tensile testing was 20. In the data provided by the authors, the number of samples was 22 (SER) and 27 (WT).
It can be assumed that only 1 or 2 silks strands from each cocoon are used for testing. Due to the obvious variance in the mechanical properties of silk, this method does not seem to accurately reflect the overall silk properties.
11. Generally, a length of 100 mm and a tensile speed of 100 mm/min are applied for mechanical performance test. It is obvious the length and tensile speed may affect the mechanical performance of silk fiber. Could the authors please explain why did you perform the test with the parameters different from literatures?
12. The diameter and cross-sectional area of the silk may affect the stress. Hence, the authors should provide the diameter and the cross-sectional area of silk and indicate how the cross-sectional area is determined.

4) writing issues
1. The abstract should be well revised to better indicate the most important findings and the significance of this study. For example, the outer layer sericin SER3 was ectopically expressed in the PSG of the silkworm via a piggyBac-mediated transgenic approach, then secreted into the inner fibroin layer, thus generating a new fiber with sericin microsomes dispersed in fibroin fibrils. The cause and effect in the sentence "Moreover, the water solubility and stability of the fibroin-colloid in the silk glandular cavity are increased, thus significantly improving the β-sheet content of fibroin, as well as the mechanical properties, moisture absorption and moisture liberation of the silk fiber" is not valid.
2. The introduction should be well revised to clearly indicated the purpose, contents and significance of this study. I'm confused about the mechanism of the metastability of ultra-high concentration aqueous solutions of Fib-H/Fib-L/P25 polymers in SGs, or altering the ancient silk structure via innovative reprogramming of the genomes of SG cells with high survival rate and silk yield.
It is difficult to understand the relationship between the sentences "the fibril structure and function of the ancient silk fiber were greatly altered" and "This method may help address the bottleneck problems of the low survival rate and low silk yield of genetically transgenic silkworms". Also, I'm confused that the function of the ancient silk fiber was greatly altered. What is the function of the ancient silk fiber and how the function of the fiber was changed?
4. In Fig. 1c, PiggBac should be piggyBac. 12. Line 366, μl → μL 13. Line 65-67, the author mentioned the reprogramming of the genomes of SG cells, which is irrelevant to the topic of this study and should therefore be deleted.
14. The manuscript should be well proof edited by a native English speaker to polish the grammar, expression and organization and correct the typos.

Reviewer #3 (Remarks to the Author):
This manuscript describes the production of a novel type of B. mori silk fiber with different molecular compositions and fiber morphology from normal silk using a transgenic technology. The authors showed that such differences have led to better mechanical and moisture absorption properties of silk. The effects of transgenesis on silk's properties are unique and might be useful in practical applications. Therefore, this manuscript would have considerable impacts on the researchers in the field of proteins materials. However, some of the authors' conclusions are not fully supported by the data in the manuscript. This manuscript is thus not appropriate for publication in Nature Communications in the present form.
Major issues: (1) Line 72-79. The authors describe many negative aspects of previous genetic alterations of silkworms. Although there are many successful examples of genetic alterations, they seem to emphasize negative aspects of genetic engineering too much by introducing some specific examples such as expressing "cytotoxin" in PSG (ref 29). The authors should summarize previous studies in a fair position.
(2) Line 82-83. This sentence would cause misunderstanding that the present genetic engineering method is not suitable for practical applications. There are actually some trials for commercial productions of genetically engineered silks in some countries. It is also unclear how the method proposed in this manuscript can solve the bottlenecks shown here. Therefore, this sentence should be omitted or revised. Moreover, the meaning of "low survival rate" is unclear, and "low silk yield" is not observed in many cases except for some specific examples such as ref 29.
(3) Figure 1b. Sericin II (may be equal to Ser2) is reported to be major coating proteins of larval silk threads spun during the growing stages (Takasu et   (5) Line 101-103. The authors describe that "the production efficiency of cocoon silk was significantly higher than that of the wild type (WT)". But no data is shown here. Figure S2i and j respectively show total cocoon weights and the ratio of silk in the total cocoon weights including pupa. The authors should clearly show the comparison of silk and silk fibroin production between SER and WT.
(6) Line 105-106. The description "the transgenic silkworm SGs have superior production performance" is not supported by experimental data because no data on silk production is provided.
(7) Line 118-119. The authors show the percentage of sericin in silk. However, without the amounts of silk and silk fibroin, it is impossible to know whether the increase of the percentage resulted from the increase of sericin production or the decrease of fibroin production.
(8) Line 119-120. This sentence is not supported without the data of actual amounts of total silk, fibroin, and sericin. SDS-PAGE analysis is also required to show the increase of Ser3 production. Some quantitative analyses such as molecular weight analysis by SDS-PAGE or GPC are required to conclude that the mutant silk has higher alkali resistance than the WT silk.
(10) Line 216. Since the authors showed only one example of a mutant silk with changed properties in this manuscript, the word "controllable" is not appropriate here. Please delete it. (11) Figure 2e. Since the number of the deconvoluted peaks are different among samples, direct comparison might be inappropriate. More careful discussion is necessary for structural analysis. The assignment of the left-most peaks is different among samples. Please explain why.
(12) Line 250. To discuss the change of the structure of silk fibroin, using only an IR analysis is not sufficient. The interpretation of the IR spectra is not convincing as described in (12). It is preferable to combine with other methods such as X-ray analysis. If the discussion of structural changes is not essential for the conclusions, I recommend that the IR analysis results are omitted.
(13) It is preferable to show the data of silk obtained from heterozygous individuals (hybrid of SER and WT lines). If such data is intermediate between SER and WT, the conclusions will be more convincing.
Minor issues: (14) The word "piggyBac" should be written in italics.
The manuscript entitled "Ectopic expression of sericin enables efficient production of ancient silk with structural changes in silkworm" by Chen et al., reported that a transgenic method was used in which the outer layer sericin SER3 in silk is secreted into the inner fibroin layer, thus generating a new structural fiber with non-fibrous sericin microsomes dispersed in fibroin fibrils.
My positive comments are as follows: Silkworm silk is a super-long natural protein fiber with ancient structure pass thousands of years without change. The silk fiber produced by thousands of silkworm varieties around the world has almost the same composition, structure and characteristics. The authors implemented a new transgenic strategy to express water-soluble non-fibrin sericin in the posterior silk gland, thus altering the fibril structure and properties of the silk. The results provide new ideas for silk protein fiber molecular design.
I have no serious criticisms regarding results and figures. I am pleased to recommend publication if the authors could address the minor concerns listed below in a carefully revised form of this manuscript.
Response: Thank you very much for reviewing our manuscript and giving such a positive opinion. Your comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our researches. We have tried our best to improve the manuscript and have made a lot of changes which we hope meet with approval.

Major comments:
Major comment 1: How to understand the "ancient silk" used in the title and introduction? Does it mean that silk production has a long history, or that modern silk still retains the molecular structure of ancient silk, even since wild silkworm evolved into silkworm?
Response: Yes, the modern silk still retains the molecular structure of ancient silk, although it has been thousands of years since silkworm was used to produce silk. We use the word 'ancient silk' by borrowing the usage of Omenetto and Kaplan (Science, 2010) [1] on silkworm cocoon silk.
The silk fiber of silkworm cocoon has a core-shell type structure, with silk fibroin as the inner core and sericin as the outer coating. Each silk fibroin brin is composed of numerous interlocking fibroin fibrils [2]. Fibroin is the main component, accounts for > 70% of cocoon silk proteins, and is composed of Fib-H, Fib-L, and P25 proteins in a 6:6:1 molar ratio [3][4][5]. At present, more than a thousand silkworm varieties have been selected and bred, but modern silk still retain the molecular structure of wild silkworm cocoon silk.

Major comment 2:
Silk gland has excellent ability of protein synthesis and secretion. This manuscript attempts to prove the reason why silk glands are often inefficient in expressing exogenous proteins, but the description is not sufficient and the explanation of this problem needs to be strengthened.

Response:
Thank you for your constructive suggestion. Relevant descriptions have been added to the discussion of the revised version.

Original text:
No ideal solution has been described to address the bottleneck problems that commonly occur in SG target tissue transgenic silkworms, such as reduced viability, abnormal SG development and low silk yield 29,30 . The growth and development of the SGs and individual mutant silkworms in this study were normal. The weight of the cocoon shell, which reflects the protein synthesis and secretion function of the SG, exceeded that of the WT by 16.8%. The cocoon layer rate, which reflects the comprehensive production capacity of mature larvae, was 14.7% higher than that of the control ( Supplementary Fig. 2). We demonstrated that while suitable exogenous protein was expressed efficiently, the protein synthesis and secretion ability of by the silkworm SG were further improved. Table S1, Bombyx mori expressed exogenous protein with molecular weight greater than 100 kDa in its silk gland (SG), which was prone to silk gland development deformity and decreased individual survival rate, and the cocoon silk production efficiency was significantly reduced, resulting in thin layered cocoon shells 24,28-30 . Although there is no description of abnormal cocoon silk yield in other reports, the expression of foreign proteins is generally not high. The highest content of foreign proteins reported is only 1.1% of the cocoon silk weight, of which the expression in the posterior silk gland is less than 0.84% of the cocoon silk [5][6][7]. It shows that the silk gland of silkworm is a highly specialized self-silk protein expression tissue, and the function of expressing foreign proteins needs to be improved.

Revision: As shown in
The growth and development of the silkworm and SG of mutants in this study were normal. The weight of the cocoon shell, which reflects the protein synthesis and secretion function of the SG, exceeded that of the WT by 16.8%. The cocoon layer rate, which reflects the comprehensive production capacity of mature larvae, was 14.7% higher than that of the control (Fig. S2). The content of SER 3 protein in mutant cocoon silk was 4.3 times higher than that in the wild type, thus indicating that sericin SER 3 in the posterior silk gland in mutants was expressed more efficiently than in the middle silk gland in the wild type (Fig. 2). We demonstrated that while suitable exogenous protein was expressed efficiently, the protein synthesis and secretion ability of by the silkworm SG were further improved.

Major comment 3:
Are fibroin fibers the same as silk fibers? If yes, it must be consistent in the manuscript; if not, please explain. And what is the difference between silk fibroin and these two?

Response:
The fibroin fibers are different from silk fibers. The fibroin fibers refer to the fiberized fibroin component inside the silk fibers.
As shown in Figure R1, silk fiber is mainly composed of sericin in the outer layer and fibroin in the inner layer. The sericin is highly hydrophilic, which acts as an adhesive joining two fibroin filaments in order to form cocoon silk, which is also known as silk fibers. Hydrophobic fibroin has good mechanical properties and is converted into raw silk and used in the production of many types of yarns and silk fabrics. Figure R1. Hierarchical structure of B. mori silk [1]. (a) B. mori silk fiber has a core-shell type structure, with silk fibroin as the inner core and sericin as the outer coating.Each silk fibroin brin is composed of numerous interlocking fibroin fibrils. Inside thefibroin fibrils, the β-sheet nanocrystals are connected by amorphous chains to form a heteronanocomposite. β-sheet nanocrystals are composed of stacked β-sheets with peptide chains connected by hydrogen bonds in each sheet. The lattice constants of the orthogonal unit cell of β-sheet nanocrystal are a = 0.938 nm, b = 0.949 nm, and c = 0.698 nm for silkworm silk. (b) Scanning electron microscopy image of native B. mori silkworm silk. (c) Atomic force microscopy image of the fibroin fibril structure in B. mori silkworm silk with a sequence of linked segments.
Editorial note: Used with permission of the Royal Society of Chemistry, from Silkworm silk-based materials and devices generated using bio-nanotechnology. Huang, Wenwen; Kaplan, David L.; Li, Chunmei; Ling, Shengjie; Omenetto, Fiorenzo G., 47, 17, 2018; permission conveyed through Copyright Clearance Center, Inc. Response: Thank you for your correction. We have revised in the revision.Major comment 5: In Figure S1, G0 adults is 85, G1 positive broods is 15, how is the mutation rate of 21% calculated?

Response:
We have revised the Fig. S1a in the revision. The total number of microinjected eggs was 1820, and 580 larvae (G0 generation) were incubated, with a hatching rate of 32 %. A total of 85 adults were obtained from G0 generation larvae, of which 71 obtained offspring (G1 generation), and the remaining 14 did not obtain offspring. Among the 71 G1 broods, positive broods were 15, and the positive rate is 21% (Fig. S1a).

Original Fig. S1a
Revised Fig. S1a Major comment 6: The paper mentions: Figure S1 (c) transgenic mutants express red fluorescent protein RFP in the eye. What period is the eye of the silkworm? Need to explain!

Response:
The detection period has been marked in the Revised legend of Fig. S1. We use the silkworm neural specific 3×P3 promoter to regulate RFP reporter gene, which can make the silkworm eye show specific red fluorescence at 554 nm excitation wavelength. Therefore, the eyes of SER transgenic silkworm positive individuals can show specific red fluorescence at 554 nm excitation wavelength at the whole stages of larva, pupa and adult, as well as the end of embryonic development. In Fig. S1c, we detected the red fluorescence of eyes at the 3rd day of the 5th instar larvae.

Major comment 7:
What is content of the sericin SER3 of the transgenic silkworms in the cocoon silk produced?
Response: Using a classical degumming method of cocoon silk to determine the sericin content in cocoon silk, the percentage of sericin in the SER group was 40.77%, and was 7.39% higher than that in the WT group (Revised Figure 2e).
In the revision, the western blotting was used to determine the SER3 content in cocoon silk with P25 as internal reference. The results showed that the SER3 content in mutant was 4.3 times higher than that in WT group (Revised Figure 2f).

Minor comments:
1. In the second paragraph on page 6, does "micro-body" in the sentence "Using immunofluorescence, … with different micro-body sizes ( Figure 2d)" refer to "microsomes"? If yes, must be unify.
Response: Accepted. We have revised in the revision.
2. In the second paragraph on page 7, does "fibroin" in the sentence "The moisture absorption and desorption performance of fibroin showed significant improvements in the SER group" refers to fibroin fiber, or silk fibroin? Please clarify.

Response:
The "fibroin" in the sentence refers to fibroin fiber, which is silk fiber with sericin removed. We have revised in the revision.
3. Desorption (in the second paragraph on page 7), dehumidification (in the second paragraph on page 7) and moisture liberation (in the abstract, and so on), do they mean the same? If yes, it must be consistent in the manuscript; if not, please explain.
Response: Thanks for the corrections. Desorption, dehumidification and moisture liberation express the same meaning in the manuscript, which has been unified as moisture liberation. We have revised in the revision. 5. In the sentence "Our laboratory has designed the artificial coding sequence Hpl, which is similar to Fib-H…" in paragraph 4 on page 11 of the discussion section, gene name of Hpl and Fib-H should be in italics.
Response: Accepted. We have revised in the revision.
6. Materials and Methods, incomplete source information of some reagents such as lack of city and Cat number.
Response: Accepted. We have added the information in the revision. Figure 1, gene name should be in italics.

Figures legends of
Response: Accepted. We have revised in the revision.
8. Supplementary Information, in Text 2, E. coli and S. aureus should not be abbreviated. This is the first appearance of these species.
Response: Accepted. We have added the full name of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in the revision.

Reviewer #2
Innovative silkworm silk structures with higher fiber performance are in great demand. In the manuscript entitled "Ectopic expression of sericin enables efficient production of ancient silk with structural changes in silkworm", the authors Chen et al. have ectopically expressed the outer layer sericin SER3 in the PSG of the silkworm by a piggyBac-mediated transgenic approach, thus generating a new fiber with improved βsheet structure contents and mechanical properties, moisture absorption and moisture liberation properties. They found that the transgenic silkworm varieties have higher cocoon production efficiency without affecting silk gland development. Hence, they concluded it is an efficient, green method to produce new silk fibers with innovative properties via the silk gland transgenic target protein selection strategy.
The topic of this paper is very interesting. The authors have presented the observation and analysis after ectopic expression of SER3 in the PSG using TEM, FTIR etc. Several aspects of this paper -especially a more complete description and discussion of the method used and the results-could be improved including: Response: Thank you very much for reviewing our manuscript and giving such a detailed opinion. Your comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our researches. We have tried our best to improve the manuscript and have made a lot of changes which we hope meet with approval.
Major comment 1: Several previous studies have reported strategies to affect the mechanical properties of silk by overexpressing a specific protein (SER3) in the fibrin layer of silk fibers (see Refs. 23-28, provided by the authors). Although they state that SER3 is an endogenous protein specifically expressed in the MSG, but not in the PSG of the silkworm. SER3 should be considered as a foreign protein of the PSG. Therefore, it is not the first report on the use of exogenous proteins to modify the properties of silk fiber, and the results are predictable.
Response: As you pointed out, it is not the first report on the use of exogenous proteins to modify the properties of silk fiber. However, there is no report of using sericin 3 (Ser3). The Refs. 23-28 reported the exogenous proteins expressed in the silk glands: Although the efforts to express and secrete exogenous proteins in the SGs of silkworms through transgenic technology to date have many successful examples of genetic alterations. However, it is still a great challenge to greatly improve the expression efficiency of foreign proteins while maintaining the cocoon silk yield, especially to express high molecular weight proteins (~100 kDa) in the posterior silk glands [1][2][3][4][5][6][7].
As shown in Table S1, Bombyx mori expressed exogenous protein with molecular weight greater than 100 kDa in its silk glands, and were prone to silk gland development deformities, decreased survival, and a significantly diminished cocoon silk production efficiency, thus resulting in thin layered cocoon shells [1][2][3][4]. Although no description of abnormal cocoon silk yield has been provided in other reports, the expression of foreign proteins is generally not high (Table S1). The highest content of foreign proteins reported is only 1.1% of the cocoon silk weight, and the expression in the posterior silk gland is less than 0.84% of the total cocoon silk [5][6][7]. The silk gland in silkworms is a highly specialized tissue with self-silk protein expression, and the expression of foreign proteins must be improved.
In this manuscript, a high molecular weight foreign protein (recombinant SER3 protein, 142 kDa) is expressed in the posterior silk gland to modify silk fiber. The highlights: (1) No adverse effect has been observed on the vitality of transgenic mutant silkworm, the survival of SER larvae infected with bacteria under stress was higher than that of the WT (revised Fig. S2m & S2n). Furthermore, no developmental phenotypic differences were observed in the silk glands of the 5th instar larvae (revised Fig. S2f).
(2) The cocoon layer weight of SER was 116.8% of that of WT group, from 0.104 g per cocoon in WT group to 0.123 g in SER group (revised Fig. S2k). The cocoon layer rate (cocoon silk production efficiency) of the SER silkworm was 114.8% of that of the WT, from 10.64% in WT group to 12.22% in SER group (revised Fig. S2l). The content of SER3 in the mutant cocoon silk (including SER3 expressed in the middle silk gland) was 4.3 times higher than that of the WT (revised Fig. 2f). Response: Thank you for your constructive suggestion. The related analysis has been added to the appropriate position in the revised manuscript.
As shown in Figure.R2 and Fig.1a, silk fibroin is secreted from PSG as a 2.3-MDa elementary unit, consisting of six sets of a disulfide-linked heavy chain (Fib-H)-light chain (Fib-L) heterodimer and one molecule of fibrohexamerin (P25) [1,2]. The H-L dimer forms a disulfide bond between Cys172 in Fib-L and the twentieth residue from the carboxyl terminus of Fib-H (Cys-c20) [3]. One internal P25 protein and six H-L dimers form the fibroin elementary unit on the cell endoplasmic reticulum through noncovalent interactions with the NTD of Fib-H [4]. The current research shows that the deletion of Fib-H and Fib-L will lead to the inability of silk gland to secrete fibroin [5][6][7], while the deletion of P25 will not affect the formation of fibroin fiber [8], indicating that P25 is not necessary to form the basic unit of fibroin. Silk fiber is composed of sericin in the outer layer and fibroin in the inner layer. The sericin is highly hydrophilic, which is expressed in the middle silk gland (MSG), while fibroin is highly hydrophobic and expressed in the posterior silk gland (PSG) [9,10].
The ratio of the number of cysteine molecules in the amino acid residues of the recombinant SER3 protein (0.50 %) was intermediate between that of Fib-H (0.10 %) and Fib-L (1.10 %) (Table S2), which has the possibility form disulfide bonds and combine with Fib-H and other silk fibroin in the PSG. As shown in Fig. 4a, recombinant SER3 protein expressed in the PSG of the mutant is unevenly distributed in the fibroin, thus indicating that the recombinant SER3 cannot form disulfide bonds and combined to Fib-H and Fib-L in the PSG. When the silk protein of PSG enters MSG, some hydrophilic SER3 will disperse from the fibroin layer and integrate into sericin layer. Meanwhile, recombinant SER3 protein can be connected to P25 with their NTD, this NTD is same as the Fib-H (Fig.1c). Thus, P25 protein may be partially separated along with recombinant SER3 protein from the interior of fibroin and distributed to the outer layer.

Major comment 3: As the authors have shown that SER3 has cysteine residue, why did SER3 not interact with Fib-H or Fib-L via disulfide bonds instead of an independent SM formation?
Response: Thank you for your comments. As you mentioned, the recombinant SER3 has the possibility of direct binding to FIb-H or FIb-L via disulfide bonds. However, as shown in Fig. 2b and Fig. 4a, fusion protein SER3 expressed in the PSG of mutant silkworm is unevenly distributed in the fibroin and formed independent liquid minisomes (recombinant SER3 protein minisomes, SM), indicating that they polymerize more easily. This result is supported by the laser confocal micrograph of silk fiber (revised Fig. 2a) and the transmission electron micrograph of silk fiber section (revised Fig. 2c, Fig.S3a & S3b), and the fluorescence micrograph in the lumen of silk gland also supports the existence of SM (revised Fig. 4a). The possible reason is that the recombinant SER3 has strong hydrophilicity and is not easy to bind to hydrophobic Fib-H or Fib-L [3][4][5].
In the manuscript, to enhance the expression and secretion of SER3 protein by PSG cells, the Fib-H gene promoter sequence and the signal peptide were introduced upstream of the Ser3 gene sequence. The EGFP reporter gene sequence and the 333 bp base sequence at the 3' end of the Fib-H gene were connected downstream of the Ser3 gene sequence. The N-end of the fusion protein contains cysteine has the possibility of combining FIB-L. Meanwhile, the P25 can combined with the NTD of recombinant SER3 protein, like combined with the Fib-H. On the other hand, it has been reported that SER3 in silk gland or cocoon silk has the phenomenon of post-translational modification, which is easy to form dimer [5]. Our western blotting results also confirmed that the recombinant SER3 was similar to the natural SER3 and appeared mainly in the cocoon silk as a dimer (Fig. 2f).

Major comment 4: Why was P25 located between fibroin and sericin?
Response: Thank you for your comments. We found this interesting phenomenon, and the detailed mechanism is worthy of further study in the follow-up study. The related analysis has been added to the discussion in the revised manuscript.
As you know, P25 is a glycoprotein containing Asn-linked oligosaccharide chains and forms a compact structure due to intramolecular disulfide linkages but associates with the H-L complex by non-covalent interactions [1]. The force of this hydrophobic interactions is much weaker than that of covalent bonds between Fib-H and Fib-L. As shown in Fig. 1c, the recombinant protein SER3 has NTD of Fib-H, which can be connected to P25 by non-covalent interactions [2] and detach it from the fibroin elementary units.
On the other hand, the recombinant SER3 expressed in the PSG of mutant is unevenly distributed in the fibroin (Fig. 4a). When the silk protein colloid in lumen of PSG enters MSG, some hydrophilic recombinant SER3 will disperse from the fibroin layer and integrate into sericin layer, due to the gradual dehydration of protein colloid or the repulsion of fibroin molecular folding. In the process of P25 dispersing from the fibroin layer to the outer layer, it is difficult to enter the hydrophilic sericin layer due to highly hydrophobic [3], resulting in most of it staying between the fibroin layer and the sericin layer. In fact, EGFP located the recombinant SER3 in the cocoon silk and found that it was more distributed between the sericin layer and the fibroin layer (revised Fig.  2b).

Major comment 5:
How did P25 affect the stability and structure of fibroin?
Response: Some studies have reported the effect of P25 protein on the stability and structure of silk fibroin fiber [1,2], so we did not describe in the manuscript. In this manuscript, there is no direct result about the P25 affects the stability and structure of fibroin.
An analysis of the 4th-instar larval silk and the cocoon silk by LC-MS/MS revealed that P25 protein is the main reason for the enhancement of mechanical properties of IV-E silk [6]. A result of recent research shows that P25 is dispensable for silk formation, it contributes to the stability of fibroin complexes during intracellular transport and affects the morphology of fibroin secretory globules in the PSG lumen [2].

Major comment 6:
The authors have shown that SM is free or independent from fibroin. SER3 is predicted to have α-helix structure without β-sheet. How did SER3 expression result in an increase in β-sheet content of silk fiber?
Response: Thank you for your comments. According to your major comment 13, we used the same peak number for curve fitting and secondary structure determination of FITR data in original Fig. 2. The results showed that the β-sheet content was 47.69% in the WT group and 47.88% in the SER3 group. The difference between the two groups did not appear to be significant. In the revised version, the FITR analysis data and expression have been modified, and the FITR results of the original Fig. 2e & 2f have been moved to supplementary information.

Major comment 7:
Usually, the amorphous region formed by α-helix and random coil structure determines the elasticity and strain of animal silk. It is doubtful that the decrease in α-helix content will not affect the strain (Figure 3c).

Response:
The mechanical properties of protein fiber and its α-Helix and β-Sheets are not completely linear. The stress of Spider silk is higher than Bombyx mori, but its α-helix content and β-sheets content is lower than silkworm [1].
In this manuscript, the addition of recombinant SER3 changes the protein composition and structure of silk fibroin microfibrils, the crystallinity of silk fibers increased after investigated by X-ray diffraction, which may be the basis for changing its mechanical properties. Response: Thank you for your comments. We have revised the related description in the revision.
As you pointed out, the sericin microsomes changed the original structure of wild type silk, resulting in the distribution of part of P25 protein on the periphery of fibroin (Fig. 2c). The current research shows that the deletion of Fib-H and Fib-L will lead to the inability of silk gland to secrete fibroin [1][2][3], while the deletion of P25 will not affect the formation of fibroin fiber [4][5].
The colloidal sericin microsomes are distributed between the fibrotic fibrils of the cocoon silk, which will change the arrangement gap of the fibrils and the sliding performance of the fibrils, but will not disrupt the fibrils.
The change of moisture absorption and moisture liberation of cocoon silk is related to the sericin existing between the fibrils.
(1) Sericin is rich in serine and aspartic acid, and has strong moisture absorption and liberation properties due to more hydrophilic groups in the side chain [6][7]. As shown in Fig. 2b and Fig.4a, fibroin contains a certain amount of sericin (recombinant SER3), which helps to improve the moisture absorption and liberation of the silk fibers.
(2) As shown in revised Fig. 2b, the recombinant SER3 in the cocoon silk was more distributed between the sericin layer and the fibroin layer. Hence, the sericin microsomes on the superficial surface of silk fibrils may fall off by degum of cocoon silk and leave tiny gaps between fibrils, thus affecting the moisture absorption and desorption properties of silk.
The Original: Moreover, the sericin microsomes dispersed in the fibrils significantly improved the moisture absorption and liberation of the silk fibers, thereby improving the performance of the textile material.
Revise: Moreover, in view of the improved moisture absorption and liberation of the silk fibers, thereby improving the performance of the textile material.
Response: Thank you for your comments. In the revised manuscript, the confocal results showed that the recombinant SER3 protein was unevenly distributed in the fibrils of the cocoon silk in the form of microsomes (Revised Fig. 2b). WB results showed that the content of SER3 relative to P25 protein in the mutant silk fiber was 4.3 times higher than that in WT group (Revised Fig. 2f). These results further support the existence of microsomes in silk fibers observed in revised Fig. 2c and Fig.S3a Response: Thank you for your comments. We have added an image of silk fiber under white light in the revision. Figure 2c, it is undoubtful that the sericin content in silk layer increased as SER3 was overexpressed in the PSG and then secreted into silk fiber. The authors should measure the expression of SER3 in the PSG and the content of SER3, Fib-H, Fib-L and P25 in the silk fiber to better support the claim of high silk yield. However, we also noticed that there is no significant difference in cocoon weight ( Figure S2i) and an increase in the cocoon layer ratio (Figure S2j), which indicated a decrease in the pupal weight of SER3 silkworm. Hence, the high silk yield via ectopic expression of SER3 in the PSG is not convincing.

Major comment 11: In
Response: Thank you for your comments. The expression of recombinant Ser3 gene was measured and reported in Fig. 4e. In the revision, the western blotting was used to determine the SER3 content in cocoon silk fiber with P25 as internal reference (Fig. 2f).
As you pointed out that there is no significant difference in cocoon weight between the mutant (1.002±0.067) and WT (0.982±0.070) (Fig.S2i). However, the PSG/SG parameter representing the development of the posterior silk gland in the SER group (Fig. S2c) was higher than that in the WT group, thus suggesting a potential advantage in the accumulation of silk material in the posterior silk gland of SER during the larval stage. The cocoon layer weight of SER was 116.8% of that of WT group, from 0.104 g per cocoon in WT group to 0.123 g in SER group (revised Fig. S2k). The cocoon layer rate (cocoon silk production efficiency) of the SER silkworm was 114.8% of that of the WT, from 10.64% in WT group to 12.22% in SER group (revised Fig. S2l). Meanwhile, the pupal weight of SER group was 0.880g (0.880±0.062) per pupa in WT group decreased to 0.877g (0.877±0.061) in SER group, no significant difference (revised Fig.  S2j). Major comment 12: In Figure 2c, the authors stated that the percentage of sericin in cocoon silk in the SER group was 7.39% higher than that in the WT group, an increase in 21.8%. How did the authors calculate the percentage of sericin in cocoon in SER3 group and make the comparison with WT group? I suggest they perform SDS-PAGE and western blot to clearly show the expression of SER3 in the fibroin layer, but not the actual yield of silk protein.
Response: Thank you for your comments. We have revised in the revision.
Using a classical degumming method of cocoon silk to determine the sericin content, the percentage of sericin in the SER group was 40.77%, and was 7.39% higher than that of 33.38% in the WT group (Revised Fig. 2e).
In the revision, the western blotting was used to determine the SER3 content in cocoon silk with P25 as internal reference. The results showed that the SER3 content in mutant was 4.3 times higher than that in WT group (Revised Fig. 2f).

Major comment 13:
In Figure 2e-f, the result is not convincing as different peak numbers were applied for the curve-fitting and the determination of the secondary structure. Also, the same wavenumber was assigned to different secondary structure. In fact, according to the data provided by the authors, the curve-fitting results using a general procedure showed that the β-sheet content was 42.05% in the WT group and 44.38% in the SER3 group. The difference between the two groups did not appear to be significant.
Response: Thank you for your correction. According to your suggestion, we used a same peak number for curve fitting and secondary structure determination of FITR data. The results showed that the β-sheet content was 47.69% in the WT group and 47.88% in the SER3 group. The difference between the two groups did not appear to be significant.
In order to analyze the microstructure of mutant silk more comprehensively, we performed SAXS and WAXD for characterizing silk crystal structure and size. The results showed that the crystallinity of fibroin fibers in SER group was increased and the mutant and WT cocoon silk have different electron density in the crystalline and amorphous regions of the periodic structure (e.g. fibroin fibrils) at the nano scale In the revised version, the FITR analysis data has been replaced by SAXS and WAXS results, and the FITR results of the original Fig. 2e & 2f have been moved to supplementary information.

Major comment 14:
The authors should provide more evidence that the silk structure has been indeed changed. I suggest to perform SAXS or WAXS for characterizing silk crystal structure and size.
Response: Thank you for your constructive suggestion. We have performed SAXS and WAXD and the related results and analysis have been added to the appropriate position in the revised manuscript.
In the revision, a D8 Advance X-ray diffractometer was used to identify the crystalline phase in the fibroin fibrils samples. According to the crystal peak position of cocoon silk, the crystal diffraction peaks of silk fibroin fibers were detected at approximately 9.0°, 20.4° and 29.1° (Fig.3k). The calculated relative crystallinity results showed that the crystallinity of fibroin fibers in WT and SER groups was 36.62% and 42.29% respectively (Fig.3l), thus indicating greater crystallinity of fibroin fibers in the SER group.
SAXS test results revealed two-dimensional images close to a double wedge shape (Fig.3m), in which the short diameter in the SER group was longer than that in the WT group, thus indicating that both SER and WT cocoon silk fibers are anisotropic, but the electron density changes before and after X-ray transmission of the two materials differed. The scattering intensity curve showed a significant difference in discrete intensity in the angle range of angle 0.1° -0.6° (Fig.3n), thus indicating that the mutant and WT cocoon silk differed in electron density in the crystalline and amorphous regions of the periodic structure (e.g., fibroin fibrils) at the nanoscale. Figure 4c, it is strange that Fib-H/L/P25 appeared in MP. I suggest the authors should take care the part of silk gland for PCR. Also, there is a significant difference between the relative expression of EGFP and SER3. Could the authors explain the difference in the expression of EGFP and SER3 since they are fused together?

Response:
The silk gland (SG) of silkworm is divided into anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG) according to different morphology and function. Previous studies have suggested that the proteins of fibroin and sericin, which are produced in the PSG and MSG, respectively [1]. The sericin gene Ser3 is naturally expressed in the anterior section of MSG of 5th instar larvae [2,3], while Fib-H is mainly expressed in the PSG [1,3]. However, so far, there is not strictly identified the expression regions of a variety of silk fibroin and sericin in SG.
In this study, according to the schematic diagram of Fig. 4a, MSG was divided into three regions, the anterior section of MSG (MA), the middle section of MSG (MM) and the posterior section of MSG (MP) respectively; PSG was divided into two regions, the anterior section of PSG (PA) and the posterior section of PSG (PP). When PCR testing, we strictly sampled the intermediate area of MA, MM, MP, PA, or PP to avoid interference from adjacent silk gland tissue. Our results showed that the fibroin genes Fib-H, Fib-L, and P25 were not only expresses high in the entire PSG (PA and PP) of WT silkworm, but also has a slight expression in MP (Fig. 4c). The Fib-H/L/P25 appeared in MP can be verified in multiple silkworm varieties, which is a reliable result. Hence, EGFP and Ser3 genes driven by Fib-H promoter were not only expresses high in PA and PP of SER silkworm, but also has a slight expression in MP (Fig 4c, 4d &  4e).
Using different primers to PCR a gene from the same sample, the electrophoretic bands of PCR products will be different due to the difference of optimal PCR conditions. The EGFP and Ser3 in MP are transcripts of a fusion genes, there is no difference (or minimal difference) in the copy number of mRNA in theory, but their PCR primers are different, the optimal PCR conditions are different, and it is normal for the electrophoresis bands of PCR products to differ. According to the results of Fig. 4c-4e, the recombinant gene EGFP and Ser3 driven by Fib-H promoter were not be expressed in MA and MP, but were expressed in PP, PA and MP. Meanwhile the expression level of the EGFP and Ser3 both decreased successively in PP-PA-MP, and the expression trend was the same as Fib-H gene. Major comment 16: According to the results provided by the authors, the silk structure of the mutant was changed dramatically. Then the author should analyze the microstructure of mutant silk more comprehensively, as FTIR is usually considered as a semi-quantitative method. Wide-angle X-ray diffraction or small-angle X-ray scattering could clearly characterize the crystallinity, grain size, orientation and other key structural information of polymer materials, which should be supplemented to better indicate the structural changes of mutant silk.
Response: Thank you for your comments. The SAXS and WAXD was performed for characterizing silk crystal structure and size.
In the revision, a D8 Advance X-ray diffractometer was used to identify the crystalline phase in the fibroin fibrils samples. According to the crystal peak position of cocoon silk, the crystal diffraction peaks of silk fibroin fibers were detected at approximately 9.0°, 20.4° and 29.1° (Fig.3k). The calculated relative crystallinity results showed that the crystallinity of fibroin fibers in WT and SER groups was 36.62% and 42.29% respectively (Fig.3l), thus indicating greater crystallinity of fibroin fibers in the SER group.
SAXS test results revealed two-dimensional images close to a double wedge shape (Fig.3m), in which the short diameter in the SER group was longer than that in the WT group, thus indicating that both SER and WT cocoon silk fibers are anisotropic, but the electron density changes before and after X-ray transmission of the two materials differed. The scattering intensity curve showed a significant difference in discrete intensity in the angle range of angle 0.1° -0.6° (Fig.3n), thus indicating that the mutant and WT cocoon silk differed in electron density in the crystalline and amorphous regions of the periodic structure (e.g., fibroin fibrils) at the nanoscale.
Major comment 17: It is suggested that proteomic analysis of degumming mutant silk should be performed to precisely determine SER3 content in silk fiber, which is essential to address the mechanism by which SER3 affects the structure and properties of silk.
Response: Thank you for your suggestion. In the revision, we do not provide proteomic data, the western blotting was used to determine the SER3 content in cocoon silk with P25 as internal reference. The results showed that the SER3 content in mutant was 4.3 times higher than that in WT group (Revised Fig. 2f). As shown in Fig.S3, the laser confocal microscope clearly observed that the EGFP labeled recombinant SER3 protein was still widely distributed in the degummed silk fibers. The results in Fig. 2b revision showed more clearly that the recombinant SER3 protein appeared in the fibroin area as particles of different sizes. The measurable particle size was 0.05-0.50 μm, which was unevenly distributed, and the most distribution was between the fibroin layer and sericin layer, secondly is among the fibrils of silk fibroin.

Major comment 18:
The author mentioned that the number of cocoons for tensile testing was 20. In the data provided by the authors, the number of samples was 22 (SER) and 27 (WT). It can be assumed that only 1 or 2 silks strands from each cocoon are used for testing. Due to the obvious variance in the mechanical properties of silk, this method does not seem to accurately reflect the overall silk properties.

Response:
We are very sorry that is not clearly described in the method, which makes you confused. The method has been modified in the revised manuscript.
As you indicate, the mechanical properties of silk fibers in different parts of a cocoon are quite different [1]. In order to reduce this effect, the mechanical properties of raw silk are determined according to the national standard of China [GB/T 1798-2008 Testing method for raw silk]. Cocoons (20 cocoons of WT or SER) were boiled in water and fully expanded before reeling to obtain raw silk. The reeling cocoon number per raw silk is 10 cocoons, and the reeling wire speed is 44-46 m/min. The obtained raw silk fiber retained most of sericin, and one sample was taken every 3 meters approximately between 100-200 meter to determine the mechanical properties or diameter of the silk. 22 samples were measured to determine the mechanical properties in SER group and 27 samples were measured in WT group.
Major comment 19: Generally, a length of 100 mm and a tensile speed of 100 mm/min are applied for mechanical performance test. It is obvious the length and tensile speed may affect the mechanical performance of silk fiber. Could the authors please explain why did you perform the test with the parameters different from literatures?
Response: As you pointed out that the length and tensile speed may affect the mechanical performance of silk fiber. Different parameters are used in different studies [1,2,3].
In this study, an initial length of 250 mm and a tensile speed of 250 mm/min are applied for mechanical performance test, which is referred the method of Wang et al. (2009). Since the mechanical properties of silk fibers in this paper are only used for the comparison of relative values between SER and WT groups, the parameters obtained by using the same fiber length and tensile speed will not affect the comparison results. Major comment 20: The diameter and cross-sectional area of the silk may affect the stress. Hence, the authors should provide the diameter and the cross-sectional area of silk and indicate how the cross-sectional area is determined.
Response: As you pointed out, the diameter and cross-sectional area of the cocoon silk may affect the stress. However, the stress is obviously affected by the fiber structure and protein composition of cocoon wires. Fig. S3g & S3h are diameter and cross-sectional area of the silk. The diameter of the silk sample was measured using a digital microscope at 1000× magnification; multiple measurements were obtained from each sample, and the average diameter was calculated, then obtained the cross-sectional area (S = π(d/2) 2 ).

4) writing issues
1. The abstract should be well revised to better indicate the most important findings and the significance of this study. For example, the outer layer sericin SER3 was ectopically expressed in the PSG of the silkworm via a piggyBac-mediated transgenic approach, then secreted into the inner fibroin layer, thus generating a new fiber with sericin microsomes dispersed in fibroin fibrils. The cause and effect in the sentence "Moreover, the water solubility and stability of the fibroin-colloid in the silk glandular cavity are increased, thus significantly improving the β-sheet content of fibroin, as well as the mechanical properties, moisture absorption and moisture liberation of the silk fiber" is not valid.
Response: Thanks for your correction. We have made some changes to the abstract in the revision.
2. The introduction should be well revised to clearly indicated the purpose, contents and significance of this study. I'm confused about the mechanism of the metastability of ultra-high concentration aqueous solutions of Fib-H/Fib-L/P25 polymers in SGs, or altering the ancient silk structure via innovative reprogramming of the genomes of SG cells with high survival rate and silk yield. It is difficult to understand the relationship between the sentences "the fibril structure and function of the ancient silk fiber were greatly altered" and "This method may help address the bottleneck problems of the low survival rate and low silk yield of genetically transgenic silkworms". Also, I' m confused that the function of the ancient silk fiber was greatly altered. What is the function of the ancient silk fiber and how the function of the fiber was changed?
Response: Thank you for your comments. We have made some changes to the introduction in the revision.
The modern silk still retains the molecular structure of ancient silk, although it has been thousands of years since silkworm was used to produce silk. We use the word 'ancient silk' by borrowing the usage of Omenetto and Kaplan (Science, 2010) [1] on silkworm cocoon silk.
At present, more than a thousand silkworm varieties have been selected and bred, but modern silk still retain the molecular structure of wild silkworm cocoon silk. The silk fiber of silkworm cocoon has a core-shell type structure, with silk fibroin as the inner core and sericin as the outer coating. Each silk fibroin brin is composed of numerous interlocking fibroin fibrils [2]. Fibroin is the main component, accounts for > 70% of cocoon silk proteins, and is composed of Fib-H, Fib-L, and P25 proteins in a 6:6:1 molar ratio [1][2][3][4][5].
This manuscript describes the production of a novel type of B. mori silk fiber with different molecular compositions and fiber morphology from normal silk using a transgenic technology. The authors showed that such differences have led to better mechanical and moisture absorption properties of silk. The effects of transgenesis on silk's properties are unique and might be useful in practical applications. Therefore, this manuscript would have considerable impacts on the researchers in the field of proteins materials. However, some of the authors' conclusions are not fully supported by the data in the manuscript. This manuscript is thus not appropriate for publication in Nature Communications in the present form.
Response: Thank you very much for your comments concerning our manuscript. Those comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our researches. We have tried our best to improve the manuscript and have made a lot of changes which we hope meet with approval.

Major issues:
Major comment 1: Line 72-79. The authors describe many negative aspects of previous genetic alterations of silkworms. Although there are many successful examples of genetic alterations, they seem to emphasize negative aspects of genetic engineering too much by introducing some specific examples such as expressing "cytotoxin" in PSG (Ref 29). The authors should summarize previous studies in a fair position.
Response: Thanks for your comments. We have revised in the revision. As shown in Table S1, Bombyx mori expressed exogenous protein with molecular weight greater than 100 kDa in its silk gland, which was prone to silk gland development deformity and decreased individual survival rate, and the cocoon silk production efficiency was significantly reduced, resulting in thin layered cocoon shells [1][2][3][4]. In the existing reports that though the cocoon silk yield is not abnormal, the expression of foreign proteins is generally not high. The highest content of foreign proteins reported is only 1.1% of the cocoon silk weight, of which the expression in the posterior silk gland is less than 0.84% of the cocoon silk [5][6][7]. It shows that the silk gland of silkworm is a highly specialized self-silk protein expression tissue, and the function of expressing foreign proteins needs to be improved. Fig. S2. The mutant SER silkworm growth and cocoon silk production efficiency. (c) Development of the posterior silk gland. After the 5th instar larvae were given mulberry for the first time (0 h), ten larvae of the same sex (male) were randomly selected every 24 h. Complete silk glands were dissected and weighed to calculate the ratio of posterior silk gland weight to silk gland weight (PSG/SG). (i) Cocoon weight, (j) Pupal weight, (k) Cocoon layer weight, and (l) Cocoon layer rate. At 72 h after cocooning, 31 cocoons of the same sex (female) were randomly selected and weighed to calculate the percentage of cocoon shell weight in the cocoon weight (cocoon layer rate).
Major comment 6: Line 105-106. The description "the transgenic silkworm SGs have superior production performance" is not supported by experimental data because no data on silk production is provided.
Response: As shown in revised Fig. S2, there is no significant difference in cocoon weight between the mutant (1.002±0.067) and WT (0.982±0.070) (Fig.S2i). However, the PSG/SG parameter representing the development of the posterior silk gland in the SER group (Fig. S2c) was higher than that in the WT group, thus suggesting a potential advantage in the accumulation of silk material in the posterior silk gland of SER during the larval stage. The cocoon layer weight of SER was 116.8% of that of WT group, from 0.104 g per cocoon in WT group to 0.123 g in SER group (revised Fig. S2k). The cocoon layer rate (cocoon silk production efficiency) of the SER silkworm was 114.8% of that of the WT, from 10.64% in WT group to 12.22% in SER group (revised Fig.  S2l).